Tetracycline process



United States Patent TETRACYCLINE PROCESS Fred W. Tanner, Jr., Baldwin,N.Y., assignor to Chas. Pfizer 8: Co. Inc., New York, N.Y., acorporation of Delaware No Drawing. Filed Apr. 23, 1957, Ser. No.654,460 Claims. (Cl. 19580) This application is concerned with a new anduseful process for the production of tetracycline. More particularly, itis concerned with a process by which tetracycline. is preferentiallyproduced by the addition of a chemical agent to a fermentation mediumwhich in the absence of this agent produces a major proportion of oreven only chlortetracycline.

Tetracycline is a broad spectrum antibiotic the therapeutic eflicacy ofwhich has been attested by a large number of. medical inventigators asreported in many articles in the medical literature. Its. chemicalstructure was first reported in the Journal of the American ChemicalSociety, volume 74, page 4976 (1952). Its chemical relationship tochlortetracycline and oxytetracycline. is well known.

Tetracycline is generally produced by one of; two methods. Thesemethods: are catalytic dehalogenation of chlortetracycline withhydrogen, and fermentation. In both of these methods, there are definitedisadvantages. The catalytic dehalogenation of chlortetracyclinerequires that. the antibiotic be isolated from a fermentation source inrelatively high purity and then subjected to chemical action requiringexpensive processing equipment and catalysts. The fermentation method,although economically more practical, produces not only tetracycline butalso chlortetracycline; In most media the latter is generally produced.preferentially. This necessitates expensive purification procedurestoseparate the two antibiotics;

Because of its high activity and because of its high stability incertain media, tetracycline is preferred in many applications over otherantibiotics. It is apparent, therefore, that any process which means itpossible to produce tetracycline substantially uncontaminated bychlortetracycline or without the necessity of chemical processingfulfills adefinite and long-felt need in the art.

The usual tetracycline-producing microorganisms be long to the genusStreptomyces. The particular strains which are usually used areStreptomycesaureofzrciens, for example, Strepzomyces aureofaciens- (NRRL2209) and Strepzomyces viridifaciens, for example, Streptomycesvirz'difaciens (ATCC 11989). Other useful strains of Streptomycesincludethose deposited in public culture collections with-the code numbersNRRL.-B-1286, B-1287, Bl288 and ATCC 11652, 11653 and 11654. Thesemicroorganisms produce fermentation products containing substantiallyonly chlortetracycline Many attempts have been made to-alter the usefulfermentation media in order to suppress chlortetracycline in favor oftetracycline in the produced antibiotic mixture. Two of these aredescribed in the patent literature. US. Patent 2,739,924 issued to Leinand G'ourevitch describes the addition of a bromide and the like, forexample, sodium bromide to fermentation media. U.S. Patent. 2,734,018.issued to Minieri et a1. describes the use of a dechlorinatedfermentation media. While both of the described processes are capable ofproducing broths of increased tetracycline content, neither of them iscompletely satisfactory inthat. the. amount of chlortetracyclineproducedis. still relatively high. Furthermore, the dechlorination procedure islaborious and expensive.

It has now most unexpectedly been discovered that the addition ofN-cyclohexyl-Z-benzothiazole-sulfenamide surprisingly makes it possibleto produce tetracycline and chlortetracycline in mixtures containing ashigh as 98% tetracycline or even higher. N-cy clohexyl-Z-benzothi--azole-sulfenamide may be substituted on the carbon atoms of the phenylring with halogen atoms or with such groups as lower alkyl, hydroxyl,nitro, mercapto and others, but the unsubstituted compound is highlyactive per se. Other variations in the structure of the basic moleculemay be made without seriously interfering with the desirable activity.This compound is available from the Monsanto Chemical Co., under thename Santocure.

In practicing the instant invention a N-cyclohex'yl-Z-benzothiazole-sulfenamide is added to a fermentation broth of atetracycline-producing microorganism of the genus Streptomyces andaerobic fermentation is allowed to continue in the normal Way. Thefermentation may be carried out in accordance with either the well knownsubmerged or surface techniques. When the antibiotic activity of thefermentation broth is isolated, for example, by extraction and theantibiotic content analyzed, it is found that the amount of.tetracycline produced is substantially higher than. is produced in theabsence of- N-' cyclohexyl 2-benzothiazole-sulfenamide. So surprisinglyefiicient isthe process of this invention that concentrations of thetetracycline-producing agent as low as twenty parts per million producebroths containing up to 200 ,ug./ml. of tetracycline of greater thanpurity. The activity and purity of the tetracycline produced wereestablished by chromatographic methods in which known solutions oftetracycline and chlortetracyclin'e and mixtures of these were used asreference standards.

Control experiments run under exactly the same conditions and evaluatedin exactly the same produced broths containing 200 agJml. ofchlortetracycline and only a trace of tetracycline. Similarly highratios of tetracycline to chlortetracycline are achieved with:concentrations of N-cyclohexyl-2-benzothiazole-sulfenamide as low as 0.2part per million or even lower. With mostchlortetra cyclineproducing.microorganisms concentrations of about twenty parts permillion are entirely adequate but the optimum concentration may varysomewhat with the particular strain or mutant of microorganism beingused. Thus certain microorganisms may yield'optimum amounts oftetracycline at concentrations higher or lower than twenty parts permillion. It is-conceivable that with some particular strainsior mutantsthe toxicity level may be higher or lower than Withtlie microorganismsheretofore tested. The optimum concentration may depend in some casesonwhetherthe N cyclohexyl-2-benzothiazolesulfenamide is added before orafter inoculation of the fermentation media. For most applications,however, concentrations as high as 100 parts per million or even higherare tolerable. It is tobe noted that the desirable effect of obtainingextremely high tetracycline to chlortetracycline ratio in the producedantibiotic is achieved without the necessity of expensive dechlorinationproceduresbeing applied to the fermentation media.

Manysuitable fermentation media oftlie type generally used in the.production of antibioticsby fermentation methods can be used inpracticing this invention. These media will usually contain a source. ofcarbohydrates and a source of nitrogen preferably in organic form. Avariety of difi'erent metallic salts also have some value in stimulatingproduction of the. antibiotic. Among the carbohyrate solutions which areuseful are molasses, glucose,starches, glycerol, etc. Organic nitrogensources include soybean meal;- wheat gluten, peanut meal, hydrolyzate ofcasein and" other-'- p'rotein's: Certain crude antibiotic.liquondistillers solubles; yeast extract, etc. Salts such as sodiumnitrate, sodium chloride, potassium phosphate 7 materials contain growthstimnlatory substanceswhich.

are of some value in obtaining maximum yields of the These include suchsubstances as corn steep and magnesium sulfate can also be' used.Occasionally the additionofa'butfering agent such as calcium carbonateis helpful andif foaming is encountered during. 7

the growth of the organisms the addition'of oils such asflard oil,soybean oil, etc. may be advisable. Useful media are described in.U.S.Patents 2,482,055, 2,709,672

and 12,776,243 as well as in now abandoned US. patent app1ications,Serial Nos. 391,708, 391,709 and 391,710,- filed November 12, 1953.; i ag Q In practicing the instant invention the active agent may 'be addedto the fermentation media before or after inoculation. -It is, however,preferably added before significant amounts of antibiotic activity havebeen produced since prior. to the additionofN-cyclohexyl-Z-benzothia'zole-sulfenarnide the antibiotic produced ispreponderantly chlortetracycline. I

a The following examples are given solely for the purpose ofillustration only and are not to be construed as limitations of thisinvention, many apparent variations of which I are possible withoutdeparting from the spirit or scope' thereof.

V 7 Example I v v A spore suspension of Streptomyces aureofaciens wasintroduced'into 100 m1. of the following composition in a 300 ml.Erlenmeyer flask.

Sucrose Corn steep liquor 7 V 10 Ammonium'monohydrogen sulfate 2 Calciumcarbonate 2 After forty-eight hours incubationat 28 C. on a rotaryshaker, approximately 2.5 .ml. of this culture served to inoculate oneliter of a fermentation'medium of the following composition. v

rCorn starch grams per liter 20' Corn steep liquor do' 25 Ammoniumsulfate 7 5{ Magnesium sulfate hept'ahydrate ..do 2 Calcium carbonate.do '7 The medium was autoclaved forty minutes at twenty V Grams perliter having the following composition.

, I 4 Example I, and it was incubated for 100 hours at 28 C. on a rotaryshaker. Analysis of aliquot portions of the broth established atetracycline content of 200 gJml. of about 97% purity.

When this procedure was carried out in the absence ofN-cyclohexyl-Z-benzothiazolesulfenamide,' the product obtained had achlortetracycline content of more than 95%.. ll] g H .ExqmplalV A sporesuspension of, 'Stre'ptomyces aureofaciens (NRRL 2209) prepared asdescribed in Example, I was used to inoculate .100 mlof a' sterilenutrient medium v 100 ml. of tap water;

The medium was autoclaved forty minutes at twenty pounds steam.pressure. After 100 hoursincubation at 35 C. in'arotary shaker, sampleswere'with'drawn'and tested for antibiotic activity. It was found thatthe broth 100 ml. of tap water.

pounds. steam pressure. After approximately ninety hours incubation at28" Con a rotary shaker, samples of the broth were withdrawn and testedby paper chromatographic methods for antibiotic content. Iti was foundthatthe b'roth'contained 200 ,ngjml. of tetracycline and thatthisrepresented more than 98% of theltotal antibiotic ac'tivityproduced. The remainder of the antibiotic activity was attributed to'chlortetracycline.

' When this procedure was carried out in the absence ofN-yclohexyLZ-benzothiazolesulfenamide,the product ob-- tained had achlortetracycline content of more than Example II r r The procedure ofExample I, was repeatede xcept tha theN-cyclohexyl-Z-benzothiazolesulfenamide was added twenty-four hoursafter incubation and the incubation was continued for an additionalsixty-six hours at. 28" C.

Analysis of aliquot portions of the-broth established a V tetracyclinecontent of 200. g./ml. of about 95% purity.

When this procedure was carried out in the absence of'N-cyclohexyl-2-benzothiazolesulfenamide, the product.

obtained had a chlortetracycline content of more than 95%. 7 Y

V e I Examplelll .A spore suspension of Streptomycin viridifacienscontained 200 p.g./m1. of tetracycline and that this represented morethan 98% of the total antibiotic activity produced. Y r

1 When this procedure was carried out in the absence ofN-cyclohexyl-Z-benzothiazolesulfenamide, the product obtained 'had achlortetracycline content of more than 95 Example V A spore suspensionof Streptomyces ciridifaciens prepared as in Example I' was used toinoculate 100 ml. of

a sterile nutrient medium having the following composition. V

Soybean meal Q grams per liter..- 40 Sodium'pnitrate do 3N-cyclohexyl-2-benzothiazolesulfenamide, the product obtained hadachlortetracycline' content of more than 95% "Results similar to thosedescribedin Examples I, II, In,

and VWere obtained withthe following strains of Streptomyces; ATCC11652,. 11653 andv 11654 as well with NRRL, B4286, E4287 and B1288.

What is claimedis: I 1. In a' process for producing tetracycline by'aerobic fermentation with' a,chlortetracycline-producing micro organismof the genus Streptomyces,ithe improvement which comprisesrcon'ductingtheffermentation'in the presence ofN-cyclohexylr2-benzothiazole-sulfenamide.

2. In a process for producingv tetracycline by aerobic fermentation witha, chlortetracycline-producing micro-v organism of the. speciesStreptomycs aureofaciens, the

. improvement which comprises conducting the fementation in the presenceof N- cyclohexyl-2-benzothiazole-sulfenamide; a

3. In a process for producing tetracycline by aerobic fermentation withva .chlortetracycline-producing microorganism of the species Streptomyeesviridifaciens, the

' improvement which comprises conducting the fermentafenamide.

(ATCC 11989) was inoculated as described in Example 1. 51316 mediumusedwas identical with that described in tion in the presence ofN-cyelohexyl-Z-benzothiazole-sul- 4. A process as in claim wherein fromto 100 parts per million of N-cyclohexyl Z-benzothiazole-sulfen-'amideis present; e I 5. ha processof aerobic fermentation of a nutrientgrams per liter 40 r medium with microorganism of the genusStreptomyccs, said fermentation normally producing an anti-bioticconsisting substantially chlortetracycline with minor amounts oftetracycline, the improvement comprising suppressing the production ofchlortetracycline in favor of tetracyciine by the adidtion ofN-cyclohexyl-Z-benzothiazolesulfenamide to the fermentation medium.

References Cited in the file of this patent UNITED STATES PATENTS2,734,018 Mim'eri et a1 Feb. 7, 1956 2,739,924 Lein et a1. Mar. 27, 19566 FOREIGN PATENTS Switzerland Nov. 15, 1956 OTHER REFERENCES

1. IN A PROCESS FOR PRODUCING TETRACYCLINE BY AEROBIC FERMENTATION WITHA CHLORTETRACYCLINE-PRODUCING MICRO ORGANISM OF THE GENUS STREPTOMYCES,THE IMPROVEMENT WHICH COMPRISES CONDUCTING THE FERMENTATION IN THEPRESENCE OF N-CYCLOHEXYL-2-BENZOTHIAZOLE-SULFENAMIDE.